Abstract/Project Summary Neurodegenerative diseases including glaucoma are characterized by neuronal death and failure of damaged axons to regenerate. Optic nerve crush (ONC), which transects all retinal ganglion cell (RGC) axons, is often used to model this process, and to seek interventions that protect RGCs and promote regeneration. Following ONC in mice, ~80% of the RGCs die within 2 weeks, and virtually none of the survivors regenerate axons. We and others have used ONC to identify interventions that lead to increased survival, increased regeneration or both. However, these treatments are only partially effective. For example, PTEN deletion increases RGC survival by only two-fold, and of >45 RGC types, only a few (alpha-RGCs) extend axons. Additionally, the growth rates of regenerating axons are slow and, most important, the regenerating axons rarely reach their targets in the brain. It is therefore important to identify additional and improved promoters of survival and regeneration. We will address this challenge by conducting an unbiased loss-of-function screen using CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats). Based on previous work from us and others showing that knockdown-regulation of several transcription factors can improve survival, regeneration or both, we have generated an AAV2-based sgRNA library for 1629 transcription factor genes; optimized methods for delivering the sgRNAs and Cas9 to RGCs; and validated our ability to identify genes that regulate survival and regeneration. In the proposed study, we will screen the entire library to find novel repressors of programs required for neuroprotection and regeneration. For selected positive hits, we will identify RGC subtypes that are protected and/or undergo axon regeneration after individual gene knockout. Finally, as a first step to test identified candidates in a clinically relevant setting, we will choose three gRNAs with robust neuroprotective effects and test their ability to protect RGCs in a widely-used glaucoma model. We expect these studies will provide insights that will enable development of novel neuroprotective and regeneration- promoting strategies for traumatic injury and glaucoma as well as other neurodegenerative diseases.